云南省奶牛芽囊原虫的感染情况及其基因亚型分析

Blastocystis sp.infection in dairy cattle in Yunnan Province and its gene subtype analysis

目的了解云南省部分地区奶牛芽囊原虫感染情况及其基因亚型,为该病的防控提供数据支持。方法于2019年6月—2020年8月从昆明、大理、保山地区采集荷斯坦奶牛和奶水牛粪样,提取粪样DNA,巢式PCR扩增芽囊原虫小亚基(SSU)r RNA基因,测序后在NCBI上进行BLAST比对,用MEGA 6.0软件以邻接法构建系统进化树。采用SPSS 20.0与SAS 9.1软件对不同地区、季节、养殖方式、品种、性别和发育阶段奶牛感染率进行统计分析,对差异有统计学意义的因素进行二元logistic回归分析。结果共采集奶牛粪样841份,其中65份SSU r RNA基因扩增阳性,扩增片段长1100 bp。奶牛芽囊原虫总感染率为7.7%(65/841,95%CI:5.92%~9.53%),其中,昆明、保山、大理地区的感染率分别为12.9%(32/248,95%CI:8.73%~17.08%)、8.1%(19/236,95%CI:4.58%~11.52%)和3.9%(14/357,95%CI:1.91%~5.94%),不同地区之间感染率差异有统计学意义(χ^(2)=16.601,P<0.01);夏、秋、冬季的感染率分别为9.4%(56/599,95%CI:7.86%~37.30%)、8.3%(8/96,95%CI:2.80%~13.86%)和0.7%(1/146,95%CI:0~2.02%),不同季节之间感染率差异有统计学意义(χ^(2)=12.411,P<0.05);圈养、放养的感染率分别为8.3%(58/702,95%CI:6.23%~10.30%)、5.0%(7/139,95%CI:1.40%~8.67%),二者差异无统计学意义(χ^(2)=1.693,P>0.05);荷斯坦奶牛、奶水牛的感染率分别为8.4%(41/490,95%CI:5.92%~10.82%)、6.8%(24/351,95%CI:4.20%~9.48%),二者差异无统计学意义(χ^(2)=0.671,P>0.05);公牛、母牛的感染率分别为11.1%(10/90,95%CI:4.62%~17.60%)、7.3%(55/751,95%CI:5.46%~9.19%),二者差异无统计学意义(χ^(2)=1.617,P>0.05);断奶前犊牛、断奶后犊牛、育成牛、成年牛的感染率分别为2/18(95%CI:0~25.63%)、26.2%(11/42,95%CI:12.89%~39.49%)、15.1%(11/73,95%CI:6.86%~23.28%)、5.8%(41/708,95%CI:4.07%~7.51%),不同阶段间感染率差异有统计学意义(χ^(2)=29.604,P<0.01)。二元logistic回归分析结果显示,季节(P<0.05)和发育阶段(P<0.01)是奶牛感染芽囊原虫的独立影响因素。序列比对分析结果显示,1份粪样的SSU r RNA基因序列与Gen Bank登录号KX618192(ST3亚型)的序列一致性为96.3%,35份与MH104059、KC148207、MH358363(ST10亚型)的序列一致性为97.3%~99.7%,23份与MT453998、MT453999(ST12亚型)的序列一致性为98.7%~100%,6份与MH358361、MH358362、KC148205、KC148206(ST14亚型)的序列一致性为98.7%~100%。系统进化树分析结果显示,本研究中的芽囊原虫分别汇聚在ST3、ST10、ST12和ST14亚型分支上,各亚型的构成比分别为1.5%(1/65)、53.9%(35/65)、35.4%(23/65)和9.2%(6/65)。结论云南省奶牛存在芽囊原虫感染,ST3和ST12基因亚型具有人兽共患风险,应该加强对该病原体的防控。

Objective To understand the prevalence of Blastocystis sp.in dairy cattle and its gene subtype in partial areas of Yunnan Province,thereby to provide information support for the prevention and control of the protozoan infection.Methods Fecal samples were collected from Holstein cows and dairy buffalos in Kunming,Dali,and Baoshan City from June 2019 to August 2020.DNA was extracted from the fecal samples,and the small subunit(SSU)r RNA of Blastocystis sp.was amplified by nested PCR and sequenced.The sequence alignment was performed with BLAST on NCBI database.Phylogenetic trees was constructed using neighbor-joining method of MEGA6.0.SPSS 20.0 and SAS 9.1 software were used to analyze the infection rates of dairy cattle in different regions,seasons,farming patterns,breeds,genders and growth stages,and those factors with significant difference were further analysised by binary logistic regression.Results A total of 841 fecal samples were collected,of which 65 were SSU r RNA positive with the amplified fragment being 1100 bp in length.The overall infection rate of Blastocystis sp.in dairy cattle was 7.7%(65/841,95%CI:5.92%-9.53%).The infection rates were 12.9%(32/248,95%CI:8.73%-17.08%),8.1%(19/236,95%CI:4.58%-11.52%)and 3.9%(14/357,95%CI:1.91%~5.94%)in Kunming,Baoshan and Dali,with significant differences between places(χ^(2)=16.601,P<0.01);the infection rate in summer,autumn and winter was 9.4%(56/599,95%CI:7.86%-37.30%),8.3%(8/96,95%CI:2.80%-13.86%)and 0.7%(1/146,95%CI:0-2.02%),respectively,showing significant differences seasonally(χ^(2)=12.411,P<0.05);the infection rate in captive farming and field farming was 8.3%(58/702,95%CI:6.23%-10.30%)and 5.0%(7/139,95%CI:1.40%-8.67%),respectively,with no significant difference(χ^(2)=1.693,P>0.05);the infection rate among Holstein cows and dairy buffalos was 8.4%(41/490,95%CI:5.92%-10.82%)and 6.8%(24/351,95%CI:4.20%-9.48%),respectively,showing no significant difference(χ^(2)=0.671,P>0.05);the rate in bull and cow was 11.1%(10/90,95%CI:4.62%-17.60%)and 7.3%(55/751,95%CI:5.46%-9.19%),repectively,presenting no significant difference(χ^(2)=1.617,P>0.05);the rate in different growth stages of pre-weaned calves,post-weaned calves,juvenile cattle and adult cattle was 2/18(95%CI:0-25.63%),26.2%(11/42,95%CI:12.89%-39.49%),15.1%(11/73,95%CI:6.86%-23.28%)and 5.8%(41/708,95%CI:4.07%-7.51%),respectively,showing significant differences between growing stages(χ^(2)=29.604,P<0.01).Binary logistic regression analysis showed that season(P<0.05)and growth stage(P<0.01)were independent influencing factor for Blastocystis sp.infection in dairy cattle.The sequence alignment found that the SSU r RNA sequence from one fecal sample was 96.3%homologous to reference sequence Gen Bank KX618192(ST3 subtype),thirty-five samples were 97.3%-99.7%homologous to the reference sequences MH104059,KC148207 and MH358363(ST10 subtype),twenty-three samples were 98.7%-100%homologous to the reference sequence MT453998 and MT453999(ST12 subtype)and six samples were 98.7%-100%homologous to the reference sequence MH358361,MH358362,KC148205 and KC148206(ST14 subtype).Phylogenetic tree analysis showed that the SSU r RNA sequences of Blastocystis sp.were clustered at to ST3,ST10,ST12 and ST14 subtype clade,respectively,having the constituent ratio of the subtypes being 1.5%(1/65),53.9%(35/65),35.4%(23/65)and 9.2%(6/65),respectively.Conclusion Blastocystis sp.infection are found in dairy cattle in Yunnan Province,and its ST3 and ST12 subtypes may have zoonotic transmission risk,thus,it is imperative to strengthen the control and prevention of the pathogen in the province.